RNA polymerase (RNAP) is the enzyme that catalyzes the first step in gene expression--the transcription of genetic information into messenger RNA. As such it is the principal target for factors regulating gene expression and its basic functions and structural features are highly conserved evolutionarily. The broad goal of this project is the understanding of the molecular mechanism of RNAP function and regulation. In collaboration with X-ray crystallographers, Dr. Goldfarb would like to understand RNAP structure in relation to its interactions with DNA, with nucleotide substrates, and with its RNA product. Single amino acid substitutions will be generated in the cloned genes, rpoB and rpoC, which specify the two largest subunits of RNAP holoenzyme. The mutations will be characterized using a variety of in vitro assays to determine which discrete activities of the enzyme are affected. In addition, the PI proposes to develop a series of engineered nucleic acid model ligands that bind stably to RNAP to form complexes analogous to those formed during transcription elongation; these ligands will be used for co-crystallization with the enzyme to refine the structural models and ascertain differences in conformations assumed by the enzyme at discrete steps in the transcription elongation reaction.